Mass spectrometers provide a fundamental tool of experimental chemistry and have proven useful and reliable in identification of chemical and biological samples. Mass spectrometry is a technique used to determine the masses of molecules and specific fragmentation products formed following vaporization and ionization. Detailed analysis of the mass distribution of the molecule and its fragments leads to molecular identification. The combination of specific molecular identification and extreme sensitivity makes molecular spectroscopy one of the most powerful analytical tools available.
However, the typical mass spectrometer is confined to the laboratory or other fixed sites due to its relatively large size and weight, as well as its high power and cooling requirements. Thus, mass spectrometer technology has not been used as a field portable detection system. Other impediments to field use include the requirements for large amounts of fluids to collect and process samples. Field samples are often much smaller in quantity and detection of such small samples is often essential (for example, in the case of detection of a chemical or biological agent that is lethal at small doses). In addition, typical scanning mass spectrometers have high data acquisition times, which is also inconsistent with field use. Also, stationary and level mounting configurations of typical mass spectrometers are inconsistent with adaptation to field use. Rapid and frequent placement and replacement of a sample is often inconsistent with the vacuum design of the typical stationary mass spectrometer.
FIG. 1 is a schematic representation of a particular type of mass spectrometer, the linear time-of-flight (“TOF”) mass spectrometer. Pulsed ultraviolet laser 10 is used to simultaneously desorb and ionize an analyte 12 from a probe 14. The laser 10 is triggered by a digital oscilloscope 16, which simultaneously marks the time, or otherwise initiates a timer. A potential difference across an extraction region serves to accelerate the ions into a drift region (typically on the order of 1 m in length) as shown. As they pass through the drift region, the ions disperse in time, with their flight times proportional to the square root of their respective masses. An ion detector 18 at the end of the drift region records the ion signals on a digital oscilloscope 16, thus providing detection times.
If there are ions of different masses, the different flight times will give rise to a number of detection times. The trigger time and the one or more detection times thus provide one or more flight time intervals which, as noted, are related to the mass of the ion. The mass of the ion is related to the flight time interval t as follows:m=2(eV)(t/D)2where D is the drift region as shown in FIG. 1 and eV is the acceleration energy imparted by the potential difference in the extraction region.
Different masses are thus determined based on the different flight times t of the ions. The TOF mass spectrometer thus records the entire mass spectrum for every ionization event that occurs to the analyte 12. Unlike other types of mass spectrometers, a TOF mass spectrometer does not rely on a scanning mass analyzer and therefore does not experience loss of signal due to scanning. The TOF mass spectrometer is also one of the simplest chemical analyzers, comprising principally an ion source, field-free tube for a drift region, and an ion detector, as shown in FIG. 1.
In addition, the TOF mass analyzer is particularly suited to measure the mass of biomolecular ions by using matrix-assisted laser desorption/ionization (“MALDI”). With MALDI, the analyte 12 is mixed with an appropriate organic matrix, inserted into the ionization region (for example, in the region occupied by probe 14 of FIG. 1), and desorbed from the surface into the TOF drift region D. The matrix absorbs radiative energy from the laser 10 and undergoes a phase change from solid to gas. During the phase change, the analyte gains a H+ ion and is thus accelerated by the potential difference in the extraction region, in the manner described above. MALDI treatment is particularly advantageous for ionization of larger molecules because the matrix provides a buffer between the energy of the laser and the sample. This prevents the larger molecules from being broken into small fragments, where analysis of these larger fragments simplifies the identification of the analyte.
Although ions produced by MALDI can be measured on a variety of mass spectrometers, a TOF mass spectrometer is particularly qualified for MALDI applications because it has no theoretical upper mass limit. Thus, MALDI is especially suited to the desorption of the larger macromelocules required for the application of chemotaxonomic methods. Larger mass ions, such as proteins and fragments of DNA strands, are still readily processed since they only take more time to reach the detector. Consequently, both the absence of any scanning requirement and an unlimited mass range make TOF mass spectroscopy a popular method for biomolecular analysis using MALDI.
For example, recent development of TOF mass spectroscopy using MALDI has included the detection of biological weapons whose mass signatures are often found in the 10 to 100 kDa range. Another valuable application is its ability to identify peptides and proteins with very high specificity and sensitivity. This area has led to the commercial development of TOF mass spectrometers for drug development in the pharmaceutical industry. Such applications indicates that TOF mass spectrometers are also well suited for biological threat detection of mid-range toxins (on the order of 1000 to 50,000 Da) in which subfemtomole sensitivity is required.
The resolution that arises from the lack of scanning has been exploited in the laboratory for many years, and the additional advantages that arise due to the TOF mass analyzer's ability to measure the mass of biomolecular ions by using MALDI has been exploited for approximately 10 years. However, the linear TOF mass spectrometer is inconsistent with use as a field portable detection system. One problem associated with adapting a linear TOF mass spectrometer includes limitations relating to mass resolution. Mass resolution of the linear TOF mass spectrometer is expressed in time units as t/2t, where t is the total flight time and t is the peak width of each TOF mass peak in the recorded spectrum. (The peak width arises principally from a small spread of energy (eV∀Uo) imparted to ions of the same mass by the potential difference.) Therefore, assuming a constant peak width t for each ion packet (group of ions having the same mass, with the mentioned energy spread), a longer total flight time will produce a larger dispersion between ions of different masses and thus increased resolution. Accordingly, many linear TOF mass spectrometers have used long drift regions to maximize mass resolution. A long drift region, of course, is incompatible with use as a field portable detection system.
A variation of the linear TOF mass spectrometer, known as the reflector or reflectron TOF mass spectrometer, is as shown in FIG. 2. Like the mass spectrometer of FIG. 1, a laser 10 desorbs and ionizes an analyte 12, which is accelerated by the potential difference V across the extraction region and into the drift region. However, the ions travel into a reflector or reflectron region at the end of the drift region, which applies a voltage that increases linearly with distance that the ion penetrates the reflectron region (as shown in FIG. 2a). The ion reflector or reflectron generally comprises a series of equally spaced conducting rings that form a retarding/reflecting field in which the ions penetrate, slow down gradually, and reverse direction, thereby reflecting the ion's trajectory back along the incoming path, as shown in FIG. 2. Ions of a given mass pass into the reflector and are turned around at the same nominal depth within the retarding field. As shown in FIG. 2, however, the energy spread ∀Uo for ions of the same mass having a nominal energy eV results in ions having the same mass penetrating the reflector slightly more or less than the nominal depth of an ion of energy eV. Because ions having a higher energy (and velocity) penetrate deeper into the opposing field, they spend more time in the reflectron and will lag slower ions having the same mass upon exiting the reflectron. However, the lagging ions exit the reflectron at a higher velocity and thus catch up with the slower ions. Thus, instead of continuing to disperse through the drift region (as in the linear TOF mass spectrometer), the reflectron imparts a focusing effect on the ions traveling in the drift region.
For the reflectron configuration of FIG. 2, the time of flight is given by:t=(m/2 eV)exp(−1/2) [L1+L2+4d]The voltage placed on the last lens element Vr is generally slightly larger than the accelerating volgate V, so that the average penetration depth d will be slightly shorter than the reflectron depth. Using this geometry, first-order kinetic energy focusing at the detector 18 for ions having the same mass is achieved when L1+L2=4d.
Thus, the reflectron configuration tends to improve the resolution while also providing a more compact total drift region. However, the above description applies to ions formed during the laser pulse (“prompt” fragmentation), not to fragment ions formed after the laser pulse that are the product of either slow unimolecular decay or bimolecular collisions (“metastable” ions). If these late-forming fragment ions are created before they exit the extraction region, the resulting TOF mass peaks are asymmetrical in the time domain and exhibit skewed peak shapes. If, on the other hand, the metastable ions are formed during their flight through the drift region (e.g., by collision with background gas), they are called post-source decay (PSD) ions. PSD peaks in TOF mass spectrometer data are particularly prevalent among peptides (small fragments of proteins), due to their propensity to break the peptide linkage along the amino-acid backbone long after the initial acceleration. The PSD product ion peaks are thus attributable to amino-acid chain fragments of the original peptide precursor.
While detection of PSD ions can be useful in biochemical analysis due to the sequencing information they yield, detection of PSD ions can be difficult. Relying on the property that all ions acquire the same energy within the source, traditional TOF mass spectrometers function by causing dispersion of ion velocities proportional to the ions' respective masses. However, PSD product ions are formed during the drift period, thus their velocities equal that of their precursor. Hence, their energies, rather than their velocities, are dispersed in direct proportion to their masses. Under these circumstances, a linear TOF (such as that shown in FIG. 1) cannot detect the presence of product ions, since their arrival at the detector occurs simultaneously with that of their parent ions (i.e., no field gradient exists to separate the ions in time).
In addition, for the reflectron TOF mass spectrometer, the fragment of a PSD ion will retain half the initial kinetic energy of the precursor ion. Hence the fragment will penetrate only halfway into the reflector shown in FIG. 2. If the focal point has been selected so that the total TOF drift region L=L1+L2=4d, as described above, then d must be reduced by a factor of 2 for focusing of the fragment. L is consequently reduced to satisfy the focusing relationship, thus the focal point for the fragment is shifted closer to the reflector. Each PSD fragment ion (as well as the original ion) is therefore focused to a different point in space.
In several commercial TOF instruments, focusing across the entire PSD spectrum is accomplished by stepping the voltage of the reflectron using 10 to 20 reflectron segments. The reflector voltage is decreased for successive laser desorption and ionization of the analyte; thus, progressively lower mass portions of the PSD spectrum are focused as the reflector voltage is decreased. The entire spectrum is then reconstituted by “stitching” together the individual spectral fragments, in effect, constructing a unified spectrum using the successive segments. This brute-force method of acquiring PSD spectra has the effect of converting the TOF mass spectrometer into a scanning instrument. This defeats a primary strength of the TOF mass spectrometer, namely the ability to rapidly acquire a complete mass spectrum without the need for any type of scanning procedure. As a result, precious sample may be consumed by the laser desorption process during the time required for the reflectron scanning process. Calibration is also difficult since each segment of the PSD spectrum corresponds to a different calibration curve. Additional power is also consumed.
A TOF mass spectrometer having a reflectron with an electric field determined by the equation for a circle, as shown in FIG. 2b provides focal points that are considerably closer to one another, thus enabling the recording of ions (as well as PSD fragments of ions) over the entire mass range at high resolution from a detector located at one position in the focal region. This electric field may be accomplished by tailoring the voltages to the plates comprising the reflectron so that the voltage magnitudes for successive plates increase in accordance with the equation of a circle. Further details of such a nonlinear reflectron TOF mass spectrometer is described in U.S. Pat. No. 5,464,985 to Cornish et al., entitled “Non-linear Field Reflectron”, issued Nov. 7, 1995, the contents of which are hereby incorporated by reference.
Existing methods for association of mass spectral peaks with threat substances either rely on amplitudes of mass spectral peaks, which is non-specific when using the matrix assisted laser desorption (MALDI) approach, employ heuristic rules, or use knowledge networks that can consume significant set-up and computation time. Additionally existing approaches do not utilize robust peak detection algorithms for real-time, highly accurate decomposition of a continuous spectrum into a bi-valued “peaks present” spectrum.
Such existing methods include an earlier rule-based system proposed by S. Hayek and W. Doss of Johns Hopkins University Applied Physics Laboratory (JHU/APL), a “Bayesian Belief Network” approach suggested by A. Feldman and J. Lin of (JHU/APL), a “Weighted Training Set Classification” approach suggested by N. Beagley, K. Wahl, S. Wunschel, K. Jarman of Pacific Northwest National Laboratory, and a “Hyperspace Feature Vector Projection” approach suggested by T. Falcone of Alphatech.
One difficulty with both a linear and nonlinear reflectron TOF mass spectrometer is their use with ions having a relatively large mass. All ions lose some of their velocity in the reflectron. Particles having a large mass have a relatively slow initial speed. These particles are relatively slow moving and lose a portion of that velocity in the reflectron. Thus, detection of these ions requires the detector have a higher sensitivity, which also requires more sampling in order to distinguish from background noise.
In addition to these particular problems that render known TOF mass spectrometers inconsistent with a field portable detection system, any attempt to adapt TOF mass spectrometers to such use would also have many of the other difficulties described above for such use of mass spectrometers in general. These include the stationary and level mounting configurations of typical designs that is inconsistent with field use, vacuum designs that are often inconsistent with the need for rapid and frequent placement and replacement of samples in field use, as well as other impediments.
In addition, there is typically an abundant sample available for analysis in TOF and other mass spectrometers located in a laboratory. Thus, a highly resolved spectrum may be achieved by repeated ionization and detection of the analyte. By contrast, in the field, only a small and diffuse sample may be available for collection from the environment. In addition, for a laboratory mass spectromenter, the samples are often prepared in a liquid state and placed in the extraction region. Because the extraction region of a typical laboratory mass spectrometer is relatively large, the small protrusion of such a liquid sample into the extraction region does not provide a substantial impact on the acceleration of the emitted ions. However, if such a liquid sample were used in a more compact extraction region of a mass spectrometer adapted for portable field use, the protrusion would affect the resulting energy imparted to the ions. In addition, liquid sample preparation in a field adapted mass spectrometer would be susceptible to freezing, spoiling, etc.